Protease phosphotase inhibitor cocktail

Detections of phospho-p44/42 mitogen-activated protein kinase (MAPK), phospho-p38 MAPK, and NF-κB p65 were performed using western blot. After PA treatment, cells lysed in buffer containing 1% Triton X-100 (Sigma-Aldrich) and Protease/Phosphotase Inhibitor Cocktail (Cell Signaling, Danvers, MA, USA). Protein concentrations assessed with Quick Start Bradford Protein Assay kit following manufacturer's protocol. Lysates loaded with Laemmli Sample Buffer onto Any kD Mini-PROTEAN-TGX pre-cast polyacrylamide gel (Bio-Rad) and separated by electrophoresis. Gels transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA) and detected with primary antibodies for MAPK (Cell Signaling) or NF-κB (Sigma-Aldrich) pathways, secondary horse radish peroxidase-conjugated antibody (Abcam, Cambridge, England), and chemiluminescent substrate (Thermo Fisher Scientific) before visualization with ChemiDoc MP System (Bio-Rad).